In collaboration with clinical investigators in NIDDK, we are continuing our analysis of the antiviral immune response of patients who have developed acute and chronic HCV infection. The aim of these studies is to compare CD4 and CD8 T cell responses in persons whose infection resolves either spontaneously or after interferon-based treatment to those who are persistently infected, to determine factors contribute to the exhaustion of HCV-specific T cell responses in persistent infection and to identify the mechanisms responsible for defective immune responses in chronic infection. In collaboration with Dr. Brian Edlin, an extramural investigator, we have completed and published the analysis of immune responses of injection drug users (IDUs) who have demonstrated a reduced risk of developing chronic re-infection despite continuing exposure to the virus. To identify immunological correlates for this apparent protection we studied HCV-specific immune responses in long-term (>10 years) IDUs. HCV-specific T cell responses were assessed in proliferation, Elispot, IFN-&#947;secretion and cytotoxicity assays, whereas HCV-specific antibodies were assessed in enzyme immunoassays (EIA), chemiluminescent assays and in vitro neutralization assays. Results: HCV-specific T-cell proliferation and IFN-&#947;production were more common in nonviremic EIA-positive (16/17 94%) than in viremic EIA-positive IDUs (9/20 45%, p=0.003) and also present in 16/26 (62%) nonviremic EIA-negative IDUs. In contrast, 19/21 (90%) viremic IDUs displayed neutralizing antibodies compared to 9/16(56%) nonviremic EIA-positive IDUs (p=0.04) and 0/24 (0%) nonviremic EIA-negative IDUs. Nonviremic IDUs with neutralizing antibodies were older (p=0.0115) than those without neutralizing antibodies, but did neither differ in IDU duration nor in HCV-specific T-cell responses. Thus, the reduced risk of HCV persistence in previously HCV-recovered IDUs correlated with T-cell responses and prolonged antigenic stimulation appears required to maintain humoral responses. In a second project with our extramural collaborator, Dr. Brian Edlin, we are currently studying the immunology of acute HCV infection in the Swan Project. The Swan Project began recruiting injection drug users aged 18-35 on the Lower East Side of Manhattan in 2005;more than 500 IDUs have been interviewed and tested for HIV and HCV antibody by our collaborators, and 150 who have tested HCV antibody-negative have enrolled in a prospective cohort. Cohort members undergo biweekly risk behavior interviews and HCV RNA testing to detect new HCV infections as soon as possible after they are acquired. We are prospectively studying the clinical features, immunology, and virology of acute infection, and comparing those who clear infection, those who develop chronic infection, and those who remain uninfected. Our study is unique in that blood specimens are collected before infection and weekly during acute infection. Participants who do not clear infection within 90 days are offered antiviral treatment through a unique multidisciplinary program. To date, we have identified 20 prospectively observed infections, 13 persons in the seronegative window period on enrollment, 3 recent seroconverters, and 10 possible reinfections. During the past year this study allowed a detailed analysis of a unique subject who spontaneously cleared HCV 1a after more than 65 weeks of infection. Spontaneous clearance of hepatitis C virus (HCV) in established chronic infection is exceedingly rare and has never been studied immunologically. Plasma and peripheral blood mononuclear cells, obtained biweekly for the first 30 weeks and at 12 additional time points between week 30 and 122 of infection, were used to measure alanine aminotransferase (ALT), viremia and cytokine/chemokine levels and to qualitatively and quantitatively characterize HCV-specific T-cell and neutralizing antibody responses. Acute hepatitis C developed 8-12 weeks after infection with ALT levels reaching 603 U/L, and a transient 1.3-log decrease in HCV titer. Vigorous, multi-specific T-cell responses emerged concomitantly, but failed to clear HCV even in the absence of viral mutations. Following the typical pattern of T-cell exhaustion, IFN-gamma secretion (in response to T-cell stimulation with HCV peptide pools) waned progressively and IP-10, IL-22 and sTNFRII levels decreased to normal levels by week 30. Unexpectedly, T-cell responses re-surged at week 48 concomitant with an ALT increase to 210 U/l and a newly evolving HCV 1a-specific neutralizing antibody (nAb) response that selected several mutations in a conformational B cell epitope. Viral titer decreased by 1 log in response to the immune response and HCV became undetectable between weeks 65 and 78 in the presence of persisting multi-specific HCV-specific T-cell responses and high nAb titers. The subject remained negative for HCV until week 122, and was deemed to have resolved infection. This unique scenario provides evidence for spontaneous reversion of T-cell exhaustion and de novo generation of effective nAb responses, resulting in clearance of chronic HCV infection.